Therapeutic and preventive agent containing dolichol

ABSTRACT

Dolichol or phosphate thereof is useful as a therapeutically treating and preventive agent, in particular, for hyperuricuria, hyperlipemia, diabetes and hepatic diseases.

The invention relates to a therapeutic treatment and preventive agentcontaining, as a pharmaceutically effective component, dolichol or aphosphate thereof. The invention agent is effective to treat andprevent, in particular, hyperuricemia, hyperlipemia, arteriosclerosis,diabetes and hepatic diseases; and then improve lipometabolism.

Dolichol is a polyprenol having the following structure and occurs inyeasts and mammals: ##STR1## wherein n represents an integer of 14 to24.

Dolichol is characterized by the presence of two trans-isoprene unitsand a cis-isoprene unit attached thereto and a saturatedalcohol-terminal (α-terminal) isoprene unit. Dolichol is supposed toplay an important role in life conservation of organisms and expected tobe available as an active ingredient for various pharmaceuticals.

Under these circumstances, we have examined the pharmaceuticalavailability of dolichol for a long time. As a result of our researches,we have found that dolichol is unexpectedly effective to treat andprevent hyperuricemia such as gout, hyperlipemia, arteriosclerosis,diabetes and hepatic diseases; and then improve lipometabolism.

Dolichol to be used in the embodiments of the present invention may beprepared by any convenient method. That is to say, it may be extracted,for example, from swine liver (cf. F. W. Burgos et al., BiochemicalJournal, 88, 470(1963)) or swine pancreas (cf. Japanese PatentApplication No. 12622/1983). Alternately, it may be prepared byfermentation with microorganisms. Furthermore it may be chemicallysynthesized.

The aforementioned chemical structure indicates that dolichol may occurin various forms depending on n. Dolichol being used in the presentinvention may be either a single compound wherein n is a particularinteger (e.g. n is 19) or a mixture of compounds wherein n representsvarious integers.

For example, dolichol originating from human liver is believed toconsist of 0.9% of the compound wherein n is 16; 8.8% of the compoundwherein n is 17; 36.6% of the compound wherein n is 18; 37.7% of thecompound wherein n is 19; 12.4% of the compound wherein n is 20; 3.2% ofthe compound wherein n is 21 and 0.7% of the compound wherein n is 22.On the other hand, dolichol originating from swine liver is believed toconsist of a small amount of the compound wherein n is 10; 2.5% of thecompound wherein n is 16; 19.8% of the compound wherein n is 17; 43.5%of the compound wherein n is 18; 28.6% of the compound wherein n is 19and 5.5% of the compound wherein n is 20. Furthermore dolicholoriginating from yeast is believed to consist of 3.0% of the compoundwherein n is 12; 14.1% of the compound wherein n is 13; 43.5 % of thecompound wherein n is 14; 34.5% of the compound wherein n is 15 and 4.8%of the compound wherein n is 16. Dolichol to be used in the embodimentsof the present invention may obviously include the dolichols asdescribed above as well as those extracted from other animal andvegetable tissues and having various compositions.

The expression "dolichol phosphate" as used herein means an ester whichis formed by bonding the terminal hydroxyl group of dolichol tophosphoric acid and has the following chemical structure; ##STR2##wherein n represents an integer of 14 to 24 and m represents an integerof 1 to 3.

Similar to free dolichol, dolichol phosphate being used in the presentinvention may be a single compound wherein n is a particular integer ora mixture of compounds wherein n represents various integers.

The therapeutic effect of the invention will be described below withreference to experimental examples. The effect on hyperuricuria is shownin Experimental Examples 1 and 2; that on hepatic diseases, in No. 3;that on diabetes, in Nos. 4 and 5; and that on hyperlipemia, in Nos. 6and 7.

EXPERIMENTAL EXAMPLE 1

60 mg/kg of Streptozotocin was injected into the tail vein of each maleSprague-Dawley rat of 140 to 160 g in body weight to thereby inducehyperuricuria experimentally. Three days after the injection ofStreptozotocin, 0.3 ml of a 1% dolichol/lecithin emulsion was injectedinto the femoral muscle of the rat once a day for four days. One dayafter the final administration, heparinized blood was collected from theaorta under etherization. The heparinized blood was centrifuged at 3,000r.p.m. for 10 min and then the uric acid in the supernatant wasdetermined. Table 1 shows the result.

                  TABLE 1                                                         ______________________________________                                        Group                n     Uric acid                                          ______________________________________                                        Normal rats not treated in the                                                                     5     1.5 ± 0.19                                      way as described in the                                                       Experimental Example 1                                                        Hyperuricemic rats treated in the                                                                  5      2.9 ± 0.85*                                    way as described in the                                                       Experimental Example 1                                                        Hyperuricuric rats administered                                                                    4     1.4 ± 0.22                                      3 mg of dolichol as described in                                              the Experimental Example 1                                                    ______________________________________                                    

EXPERIMENTAL EXAMPLE 2

50 mg/kg of Streptozotocin was injected into the tail vein of each maleSprague-Dawley rat of 170 to 190 g in body weight to thereby inducehyperuricuria experimentally. 10 days after the injection ofStreptozotocin, 0.3 ml of a 1% dolichol monophosphate/lecithin emulsionwas injected into the femoral muscle of the rat once a day for 42 days.One day after the final administration, heparinized blood was collectedfrom the aorta under etherization. The heparinized blood was centrifugedat 3,000 r.p.m. for 10 min and then uric acid in the supernatant wasdetermined.

                  TABLE 2                                                         ______________________________________                                        Group                 n     Uric acid                                         ______________________________________                                        Hyperuricemic rats treated in the                                                                   8     7.3 ± 5.4                                      way as described in the                                                       Experimental Example 2                                                        Hyperuricuric rats administered                                                                     8     1.8 ± 0.63*                                    3 mg of dolichol monophosphate                                                as described in the Experimental                                              Example 2                                                                     ______________________________________                                    

The results of the Experimental Examples 1 and 2 clearly indicate thatadministration of dolichol or its phosphate would significantly lowerhigh uric acid values in blood to a normal level. Accordingly thesecompounds are extremely effective therapeutic and/or preventive agentsfor hyperuriemia.

The compounds of the present invention may be administered for treatingand/or preventing hyperuricemia such as gout either orally orparenterally, e.g., intramuscularly, hypodermically or intravenously.

EXPERIMENTAL EXAMPLE 3 Effects of dolichol and its phosphate on hepaticregeneration after hepatectomy (1) Method

Male Sprague-Dawley rat (7 weeks of age, each 210 to 240 g in bodyweight) were subjected to hepatectomy according to the method reportedby Higgins and Anderson. That is, the rat had an abdominal operationunder etherization to remove the right and left median lobes of liver(approximately 72% on average). After suturation, the rat was fed withOriental Solid Feed and tap water in a usual manner. After a certainperiod, the rat was exsanguinated under etherization. Then the liver wasweighed to calculate the hepatic regeneration rate by the followingequation: ##EQU1## wherein the weight of residual liver was determinedby subtracting the weight of the removed liver from the weight of thewhole liver at the operation which was regarded 4.65 g per 100 g of bodyweight.

(2) Effects of dolichol on the regeneration rate

30 mg/kg of a dolichol/lecithin emulsion was administeredintraperitoneally to a rat once a day for eight consecutive days (fivedays preoperation and three days post-operation). Table 3 shows theresults

                  TABLE 3                                                         ______________________________________                                                                         Regeneration                                 Group        n     Dose (mg/kg/day)                                                                            rate (%)                                     ______________________________________                                        Control group                                                                              14    --            40.81 ± 1.97                              Dolichol-administered                                                                       7    30            48.78 ± 1.54**                            group                                                                         ______________________________________                                    

Table 3 indicates that continuous intraperitoneal administration ofdolichol of the present invention at a dose of 30 mg/kg/day wouldsignificantly raise the regeneration rate.

(3) Effect of dolichol phosphate on the regeneration rate

Three days after the hepatectomy, dolichol monophosphate wasintravenously administered to a rat followed by the evaluation afterfour days, i.e., seven days after the operation. Table 4 shows theresult.

                  TABLE 4                                                         ______________________________________                                                       Dose       Regeneration                                                                           Total protein in                           Group    n     (mg/kg/day)                                                                              rate (%) blood (mg/dl)                              ______________________________________                                        Normal rats                                                                            2     --         --       6.35                                       Control  8     --         70.5 ± 2.0                                                                          5.8 ± 0.05                              Dolichol 8     15         77.5 ± 5.2                                                                          6.1 ± 0.08*                             phosphate                                                                     ______________________________________                                    

Table 4 clearly indicates that an intravenous injection of dolicholphosphate after a posthepatectomic increase in blood cholesterol wouldraise the regeneration rate and significantly increase the total proteincontent in blood.

The result of this Experimental Example teaches that dolichol and aphosphate thereof according to the invention would accelerate therecovery of the function of hepatic cells at regeneration.

The compounds of the present invention are effective for treating and/orpreventing hepatic diseases such as inflammation, denaturation,necrosis, choleresis insufficiency and saccharometabolic disorder causedby alcohol, malnutrition, viruses, chemical substances, toxins or thelike.

Thus the compounds of the present invention are effective for treatmentand/or preventing hepatic diseases including acute and chronic hepatitisand hepatocirrhosis.

EXPERIMENTAL EXAMPLE 4

60 mg/kg of Streptozotocin was injected into the tail vein of each maleSprague-Dawley rat of 140 to 160 g in body weight to thereby inducediabetes experimentally. Three days after the injection ofStreptozotocin, 10 μl of whole blood was collected from the tail vein ofthe rat to determine the blood sugar content by the glucose oxidasemethod. The rat showed a blood sugar content of not less than 250 mg/dl,which indicated that it suffered from diabetes. Three days after theinjection of Streptozotocin, 0.3 ml of a 1% dolichol/lecithin emulsionwas injected into the femoral muscle of the rat four times a day forfour days. One month after the final administration, heparinized bloodwas collected from the aorta under etherization. The heparinized bloodwas centrifuged at 3,000 r.p.m. for 10 min and the triglycerides andglucose in the supernatant were determined.

Table 5 shows the results.

                  TABLE 5                                                         ______________________________________                                                                            Glucose                                   Group          n     Triglyceride (mg/dl)                                                                         (mg/dl)                                   ______________________________________                                        Normal rats not treated in                                                                   5     101 ± 6     184 ± 10                               the way described in the                                                      Experimental Example 4                                                        Diabetic rats treated in the                                                                 5     765 ± 277   548 ± 30                               way as described in the                                                       Experimental Example 4                                                        Diabetic rats administered                                                                   4     199 ± 54    477 ± 43                               3 mg of dolichol as des-                                                      cribed in the Experimental                                                    Example 4                                                                     ______________________________________                                    

EXPERIMENTAL EXAMPLE 5

50 mg/kg of Streptozotocin was injected into the tall vein of each maleSprague-Dawley rat of 170 to 190 g in body weight to thereby inducediabetes experimentally. One day after the injection of Streptozotocin,10 μl of whole blood was collected from the tail vein of the rat todetermine the blood sugar content by the glucose oxidase method. The ratshowed a blood sugar content of not less than 250 mg/dl, which indicatedthat it suffered from diabetes. 10 days after the injection ofStreptozotocin, 0.3 ml of a 1% dolichol monophosphate/lecithin emulsionwas injected into the femoral muscle of the rat once a day for 42 days.One day after the final administration, heparinized blood was collectedfrom the aorta under etherization. The heparinized blood was centrifugedat 3,000 r.p.m. for 10 min and then triglycerides and glucose in thesupernatant were determined.

                  TABLE 6                                                         ______________________________________                                                               Triglyceride                                                                             Glucose                                     Group           n      (mg/dl)    (mg/dl)                                     ______________________________________                                        Diabetic rats treated in                                                                      8      2160 ± 1721                                                                           650 ± 108                                the way as described in the                                                   Experimental Example 4                                                        Diabetic rats administered                                                                    8       514 ± 361*                                                                           534 ± 64*                                3 mg of dolichol monophos-                                                    phate as described in the                                                     Experimental Example 5                                                        ______________________________________                                    

The results in Experimental Examples 4 and 5 clearly indicate that theadministration of dolichol or its phosphate, i.e. the compounds of thepresent invention, significantly lower the content of both triglycerideand glucose.

Accordingly these compounds are extremely effective therapeutic and/orpreventive agents for diabetes.

EXPERIMENTAL EXAMPLE 6

60 mg/kg of Streptozotocin was injected into the tail vein of each maleSprague-Dawley rat of 140 to 160 g in body weight to thereby inducehyperlipemia experimentally. Three days after the injection ofStreptozotocin, 0.3 ml of a 1% dolichol/lecithin emulsion was injectedinto the femoral muscle of the rat once a day for four days. One monthafter the final administration, heparinized blood was collected from theaorta under etherization. The heparinized blood was centrifuged at 3,000r.p.m. for 10 min and then total protein (TP), albumin (Alb), totalcholesterol (T.CHO) and triglycerides (TG) were determined.

Table 7 shows the results.

                                      TABLE 7                                     __________________________________________________________________________                           Alb    T.CHO   TG                                      Group         n TP     (g/dl) (mg/dl) (mg/dl)                                 __________________________________________________________________________    Normal rats not treated                                                                     5 5.9 ± 0.2                                                                         3.3 ± 0.09                                                                        66 ± 14                                                                            101 ± 6                              in the way as described in                                                    the Experimental Example 6                                                    Hyperlipemic rats treated                                                                   5  5.6 ± 0.11*                                                                       2.9 ± 0.22*                                                                       172 ± 26***                                                                       765 ± 277**                          in the way as described in                                                    the Experimental Example 6                                                    Hyperlipemic rats adminis-                                                                  4 5.6 ± 0.23                                                                        3.0 ± 0.13                                                                           89 ± 11.sup.###                                                                 199 ± 54.sup.###                     tered 3 mg of dolichol as                                                     described in the                                                              in the way as described                                                       Experimental Example 6                                                        __________________________________________________________________________

EXPERIMENTAL EXAMPLE 7

50 mg/kg of Streptozotocin was injected into the tail vein of each maleSprague-Dawley rat of 170 to 190 g in body weight to thereby inducehyperlipemia experimentally. 10 days after the injection ofStreptozotocin, 0.3 ml of a 1% dolichol monophosphate/lecithin emulsionwas injected into the femoral muscle of the rat once a day for 42 days.One day after the final administration, heparinized blood was collectedfrom the aorta under etherization. The heparinized blood was centrifugedat 3,000 r.p.m. for 10 min and then total cholesterol (T.CHO),triglycerides (TG), phospholipids (PL) and non-esterified fatty acids(NEFA) in the supernatant were determined.

Table 8 shows the results.

                                      TABLE 8                                     __________________________________________________________________________                    T.CHO   TG     PL      NEFA                                   Group         n (mg/dl) (mg/dl)                                                                              (mg/dl) (mg/dl)                                __________________________________________________________________________    Hyperlipemic rats treated                                                                   8 186 ± 49                                                                           2160 ± 1721                                                                       415 ± 102                                                                          2.60 ± 1.1                          in the way as described                                                       in the Experimental                                                           Example 7                                                                     Hyperlipemic rats admini-                                                                   8   82 ± 32***                                                                       514 ± 361                                                                          211 ± 78***                                                                         0.98 ± 0.43**                     stered 3 mg of dolichol                                                       monophosphate as described                                                    in the Experimental                                                           Example 7                                                                     __________________________________________________________________________

The results in Experimental Examples 6 and 7 clearly indicate thatadministration of dolichol or its phosphate, i.e., the compounds of thepresent invention, would be effective for treating hyperlipemia.Accordingly these compounds serve as extremely effective lipometabolismimprovers and therapeutic and/or preventive agents for arteriosclerosisaccompanied with hyperlipemia.

Dolichol and its phosphate which are the compounds of the presentinvention are highly safe. Therefore it is possible to administer thesecompounds continuously, which also make the present invention veryvaluable.

For example, no deaths nor side effects were observed when 1,500 mg/kgof dolichol, i.e., the compound of the present invention was orallyadministered to SD rats of approximately 200 g in body weight.

The dose depends on the stage of the disease or the age of the patients.These compounds may be administered usually in amounts of approximately10 to 1,000 mg a day and preferably approximately 50 to 300 mg a day,without particular limitation.

The compounds of the present invention may be formulated in a well-knownmanner in the art into various forms such as tablets, granules, powders,capsules, injections and suppositories. These compounds may beformulated in a conventional manner by using conventional carriers.

For example, solid pharmaceuticals for oral administration may beprepared by adding an excipient with, if necessary, a binder, adisintegrant, a lubricant, a colorant, a corrigent or the like to thebase and then formulating the mixture into a tablet, a coated tablet,granule, powder, capsule or the like in a conventional manner.

Examples of the excipient are lactose, corn starch, white sugar,glucose, sorbitol, crystalline cellulose and silicon dioxide. Examplesof the bonder are polyvinyl alcohol, polyvinyl ether, ethylcellulose,methylcellulose, gum arabic, tragacanth, gelatin, shellac,hydroxypropylcellulose, hydroxypropylstarch and polyvinylpyrrolidone.Examples of the disintegrant are starch, agar, gelatin powder,crystalline cellulose, calcium carbonate, sodium hydrogencarbonate,calcium citrate, dextrin and pectin. Examples of the lubricant aremagnesium stearate, talc, polyethylene glycol, silica and hardenedvegetable oils. Any colorant which is pharmaceutically acceptable may beused. Examples of the corrigent are cacao powder, menthol, aromaticpowder, peppermint oil, borneol and cinnamon powder. The obtained tabletor granule may be coated with sugar, gelatin or the like if desired.

An injection may be prepared by adding desired agents such as a pHadjustor, a buffer, a stabilizer and a solubilizing agent to the baseand then formulating the mixture into a hypodermic, intramuscular orintravenous injection in a conventional manner.

Processes for preparing dolichol which is the compound of the presentinvention wil be given for reference.

PREPARATIVE EXAMPLE 1 Preparation of a pancreatic fat extract

1 kg of minced swine pancreas was stirred vigorously in 4 l of acetoneto extract oil and fat components. The acetone phase was separated toobtain 4.2 l of a liquor. Then the liquor was concentrated under heatingto obtain 500 ml of a concentrate. After cooling the concentrate, asolidified fat phase called a pancreatic fat extract (100 g) wasseparated.

PREPARATIVE EXAMPLE 2 Preparation of a pancreatic fat extract

2.5 kg of minced swine pancreas was stirred vigorously in 10 l ofethanol to extract oil and fat components. The alcoholic phase wasseparated to obtain 10.1 l of a liquor. Then the liquor was concentratedunder heating to obtain 1.5 l of a concentrate. After cooling theconcentrate, a solidified fat phase called a pancreatic fat extract (280g) was separated.

PREPARATIVE EXAMPLE 3 Preparation of a pancreatic fat extract

200 ml of water, 100 g of swine duodenum (10 g of pancreatin) and 10 mlof a 40% NaOH solution were added to 1 kg of minced swine pancreas. Thenthe mixture was thoroughly stirred at pH 8.5 and subjected to theactivating treatment which was employed in the activation of protease orin preparing pancreatin.

Subsequently 4.8 l of acetone was added to the mixture to extract oiland fat components. Then the acetone phase (5.3 ml) was separated.

The precipitate was further defatted and ground to obtain 220 g ofpancreatin.

The acetone phase was concentrated under heating to obtain 700 ml of aconcentrate. After cooling the concentrate, a solidified fat phasecalled a pancreatic fat extract (100 g) was separated. PREPARATIVEEXAMPLE 4

Preparation of a pancreatic fat extract

3 kg of minced swine pancreas was stirred vigorously in 12 l of a 30%ethanol solution (pH 3.0). Then the alcohol/aqueous phase (13 l) wasconcentrated under heating to obtain 5 l of a concentrate. After coolingthe concentrate, 250 g of a solidified pancreatic extract was obtained.

In addition, an intense insulin activity was observed when the liquidphase was administered intraperitoneally to a rat to examine its effectof lowering blood sugar.

PREPARATIVE EXAMPLE 5

1.5 kg of the pancreatic fat extract prepared in the Preparative Example1 was dissolved in 3 l of methanol. Then 1.7 kg of a 15% aqueoussolution of caustic soda was added dropwise to the solution at roomtemperature under stirring. The mixture was saponified for one hour at60° to 70° C. and then cooled to 50° C. Susequently the mixture wasextracted with 3 l of hexane. The organic phase was washed with 1 l of asolvent mixture (methanol/water 2:1) and allowed to stand at 4° C.overnight. Precipitated crystals were filtered and the filtrate wasconcentrated. Then the obtained concentrate was purified with silica gelcolumn chromatography by using n-hexane/benzene as an eluent.Consequently 85 mg of dolichol in the form of a colorless oil wasobtained. The dolichol prepared in the present Example was identifiedsince the retention time thereof in HPLC coincided with that of acommercial dolichol (Sigma Co., INC. D-4511).

HPLC:

stationary phase: nucleosil C₁₈ 7μ×25 cm,

mobile phase: a solvent mixture consisting of 520 parts of isopropylalcohol, 240 parts of methanol, 40 parts of n-hexane and 18 parts ofwater,

flow rate: 1 ml/min,

detection wavelength: 210 nm.

Now a Formulation Example in which dolichol which is the compound of thepresent invention and referred to as the base in the Example is used asan active ingredient will be given.

FORMULATION EXAMPLE Tablets

base: 10 g

silicic anhydride: 50 g

crystalline cellulose: 70 g

corn starch: 36 g

hydroxypropylcellulose: 10 g

magnesium stearate: 4 g

The mixture was formulated into tablets each weighing 180 mg in aconventional manner.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of treatingdiabetes which comprises administering to a diabetic subject requiringsuch treatment, a therapeutically effective amount of a diabetestreating composition comprising a pharmaceutical carrier and aneffective amount of at least one dolichol substance selected from thegroup consisting of compounds having the formulas ##STR3## wherein n isan integer of 14 to 24, and m is an integer of 1 to
 3. 2. A method asclaimed in claim 1, in which said substance has the formula ##STR4## 3.A method as claimed in claim 1, in which said substance has the formula##STR5##
 4. A method as claimed in claim 1, in which the amount of saidsubstance administered is from 10 to 1000 mg/day.